Isolation of Marker Compounds from Copaiba Essential Oil for Quality Evaluation of Dietary Supplements

Abstract

ABSTRACT Natural products have been used as a primary means of treating illness since ancient times, and they remain an important pool of U.S. FDA approved drugs. Approximately 33% of all current pharmaceutical medications discovered between 1981 and 2014 are either natural products or derived from natural products (Newman and Cragg 2016). The process of isolating specialized metabolites from medicinal plants has led to the development of particularly important medicines worldwide (Rakotomalala, Agard et al. 2013) such as Taxol® from Taxus brevifolia Nutt. (Taxaceae) and salicylic acid from the bark of Salix spp. This research is composed of five chapters; the first is focused on drug discovery, whereas the rest are focused on efforts to assess the quality and safety of copaiba oil, which is used as a dietary supplement in various parts of the world, including the United States. Mimosa pudica is marketed as a dietary supplement in the United States with claims that it supports the immune system. It is known that M. pudica grows with related species and has been misidentified with a closely related invasive species, M. pigra. A Brazilian study screened over 60 plants for anti-dermatophyte activity, and dichloromethane fractions from the methanolic extract of M. pigra showed the lowest MIC values (1.9 µg/mL) without DNA destruction at 10 and 50 µg/mL of cell viability of human leukocytes; however no further work was conducted to determine the specialized metabolite responsible for the activity shown. (de Morais, Scopel et al. 2017). Therefore, my research is aimed at isolating specialized metabolites that could be utilized as chemical markers to distinguish between M. pudica and M. pigra and which also have promising antifungal activity without human toxicity concerns. Powdered M. pigra leaves were processed via percolation in methanol at room temperature to create a crude extract after the removal of solvent under reduced pressure. This extract was fractionated by vacuum liquid chromatography (VLC) using reversed-phase C-18 silica with a gradient elution of methanol and water (0:1 to 1:0), resulting in 10 fractions. The crude fractions were tested against Candida albicans, and several of them showed promising anti-candida activity. The obtained fractions were subjected to repeated column chromatography over Sephadex LH-20, RP-18 silica, and normal phase silica to yield eight phenolic compounds. The isolated specialized metabolites were again tested against C. albicans to determine which were responsible for the anti-candida activity. We suggest that the chemical constituents isolated in this study could be used as chemical markers to differentiate M. pigra-based raw materials in various finished products, including dietary supplements that purportedly contain M. pudica. My second project focuses on assessing the safety and quality of copaiba essential oils. Copaiba oil use has been reported since the 16th century in Amazon traditional medicine, especially as an anti-inflammatory ingredient and for wound healing (da Silva, Puziol Pde et al. 2012, da Trindade, da Silva et al. 2018). This oil’s use continues today, and it is sold in various parts of the world, including the United States. Adulteration of copaiba oil or resin might occur because of the time-consuming extraction process and demand exceeding the supply(Barbosa, Yoshida et al. 2009). According to the literature, there are two methodscommonly used for copaiba oil adulteration: incorporating a low-cost vegetable oil such as soybean or mineral oil, or adding another inferior essential oil that has a similar density and flavor (Barbosa, Yoshida et al. 2009). Despite the importance of copaiba oil in folk medicine and its economic significance, limited studies have reported a validated GC/MS method for evaluating the quality of copaiba oil (Sousa, Brancalion et al.